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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Tartaric Acid
CAS No.: 87-69-4
Chemical Name: Tartaric Acid
Batch No.: B10X 1914
Physical Appearance: fine powder
Colour: white
Storage: at room temperature, protected from light
Solubility: water

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test System
Species/strain: healthy CBA/CaOlaHsD mice
Source: Harlan Winkelmann, 33178 Borchen, Germany
Sex: female (nulliparous and non-pregnant)
Age at the beginning of the study: 8 – 9 weeks
Number of animals:
- 5 mice / test group
- 3 mice / preliminary test

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1020)
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (lot no. 190110)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days)

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
6.25%, 12.5% and 25% (diluted with DMSO).
No. of animals per dose:
20 animals

3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.
Details on study design:
Topical Application
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application.
Topical applications were performed once daily over three consecutive days.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250μL of 3H-methyl thymidine, diluted to a working concentration of 80μCi/mL.

Preparation of Cell Suspension
The thickness of the ears of all animals was measured for a third time. Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining “auricular lymph nodes” were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H-Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: P-Phenylenediamine (CAS 106-50-3)
Statistics:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a-c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3 fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the lymph nodes of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

Results and discussion

Positive control results:
The reliability check was performed in July 2010. The reliability checks were audited by the QA unit periodically.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see below

Any other information on results incl. tables

Results

None of the three tested concentrations of the test item reached the stimulation index of 3.

The stimulation index at a concentration of 6.25% was0.8

The stimulation index at a concentration of 12.5% was1.8

The stimulation index at a concentration of 25% was1.5

 

The mean ear thickness on

day 1

day 3

day 6

for the 6.25% test group was

0.19 mm

0.18 mm

0.20 mm

for the 12.5% test group was

0.20 mm

0.19 mm

0.19 mm

for the 25% test group was

0.20 mm

0.20 mm

0.21 mm

for the negative control group was

0.20 mm

0.20 mm

0.21 mm

 

Conclusion

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

The results of radioactivity determination were supported by the means of the ear thickness per group, which showed no significant difference compared to the negative control.

 

Consequently, according to OECD 429 [3] and the criteria given in Annex I of Regulation (EC) 1272/2008 [6], the test item Tartaric Acid, as described in this report is expected to have no sensitising properties and therefore, should not be regarded as a dermal sensitiser.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information